Coordinated decrease and increase of gene expression of more than one gene using transgenic constructs

ABSTRACT

The present invention is directed to nucleic acid molecules and nucleic acid constructs, and other agents associated with simultaneous up- and down-regulation of expression of RNAs. Specifically it includes methods of simultaneously enhancing the expression of a first RNA at the same time as suppressing the expression of a second RNA. The present invention also specifically provides constructs capable of simultaneously enhancing the expression of a first RNA while at the same time suppressing the expression of a second RNA, methods for utilizing such agents and plants containing such agents. The present invention also provides other constructs including polycistronic constructs.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the sequence listing on diskette, containingthe file named “16517.265.SeqList.txt”, which is 24,734 bytes in size(measured in MS-DOS), and which was created on Sep. 24, 2003, are hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to nucleic acid molecules and nucleicacid constructs, and other agents associated with simultaneous up- anddown-regulation of expression of RNAs. Specifically it includes methodsof simultaneously enhancing the expression of a first RNA at the sametime as suppressing the expression of a second RNA using a singleconstruct. The present invention also specifically provides constructscapable of simultaneously enhancing the expression of a first RNA whileat the same time suppressing the expression of a second RNA, methods forutilizing such constructs and plants containing such constructs. Thepresent invention also provides other constructs including polycistronicconstructs.

BACKGROUND OF THE INVENTION

Many complex biochemical pathways have now been manipulated genetically,usually by suppression or over-expression of single genes. Furtherexploitation of the potential for plant genetic manipulation willrequire the coordinated manipulation of multiple genes in a pathway. Anumber of approaches have been used to combine transgenes in oneplant—including sexual crossing, retransformation, co-transformation,and the use of linked transgenes. A chimeric transgene with linkedpartial gene sequences can be used to coordinately suppress numerousplant endogenous genes. Constructs modeled on viral polyproteins can beused to simultaneously introduce multiple coding genes into plant cells(for a review, see Halpin et al., Plant Mol. Biol. 47:295–310 (2001)).

Enhancement of gene expression in plants may occur through theintroduction of extra copies of coding sequences of the genes into aplant cell or, preferably, the incorporation of extra copies of codingsequences of the gene into the plant genome. Over-expression may alsooccur through increasing the activities of the regulatory mechanismsthat regulate the expression of genes, i.e., up-regulation of the geneexpression.

Suppression of gene expression, also known as silencing of genes, inplants occurs at both the transcriptional level and post-transcriptionallevel. There are various methods for the suppression of expression ofendogenous sequences in a host cell. Such methods include, but are notlimited to, antisense suppression (Smith et al., Nature 334:724–726(1988)), co-suppression (Napoli et al., Plant Cell 2:279–289 (1989)),ribozymes (Kohler et al., J. Mol. Biol. 285:1935–1950 (1999)),combinations of sense and antisense (Waterhouse et al., Proc. Natl.Acad. Sci. USA 95:13959–13964 (1998)), promoter silencing (Park et al.,Plant J. 9(2):183–194 (1996)), and DNA binding proteins (Beerli et al.,Proc. Natl. Acad. Sci. USA 95:14628–14633 (1997); Liu et al., Proc.Natl. Acad. Sci. USA 94:5525–5530 (1998)).

Certain of these mechanisms are associated with nucleic acid homology atthe DNA or RNA level (Matzke et al., Current Opinion in Genetics andDevelopment 11:221–227 (2001)). In plants, double-stranded RNA moleculescan induce sequence-specific silencing. This phenomenon is oftenreferred to as double stranded RNA (“dsRNA”) in plants. This phenomenonhas also been reported in Caenorhabditis elegans, where thisgene-specific silencing is often referred to as RNA interference or RNAi(Fire et al., Nature 391:806–811 (1988). Others have reported thisphenomenon in plants, fungi and animals (Sharp, Genes and Development13:139–141 (1999); Matzke et al., Current Opinion in Genetics andDevelopment 11:221–227 (2001); Cogoni and Macino, Current Opinion inGenetics and Development 10:638–643 (2000); Sharp, Genes and Development15:485–490 (2001); Waterhouse et al., Proc. Natl. Acad. Sci. USA95:13959–13964 (1988); Wesley et al., Plant J. 27:581–590 (2001);Grierson, WO 98/53083). Wesley et al. reported the design and use of twovectors, pHANNIBAL and pHELLSGATE, that can be used as gene silencingvectors (Wesley et al., supra). These vectors are reported to contain anintron sequence between the sense and antisense sequences where thesense and antisense sequences correspond to a target coding sequence,5′UTR or 3′UTR. By utilizing a non-target intron between the targetsense and antisense sequences, a higher proportion of silencedtransformants were obtained (Wesley et al., supra). Another strategy ofgene silencing with dsRNA involves a hairpin construct with an intronspacer (Smith et al., Nature 407:319–320 (2000)).

Other suppression strategies include, without limitation, antisense andsense suppression. See e.g. Fillatti in PCT WO 01/14538.

A desired plant phenotype may require the expression of one gene and theconcurrent reduction of expression of another gene. Thus, there exists aneed to simultaneously over-express a polypeptide and suppress, ordown-regulate, the expression of a second polypeptide in plants using asingle transgenic construct. Moreover, there exists a need tosimultaneously suppress or down-regulate the expression of more than onepolypeptide using a single construct.

SUMMARY OF THE INVENTION

The present invention includes and provides a nucleic acid moleculecomprising a first nucleic acid segment comprising a polypeptideencoding sequence and a second nucleic acid segment comprising a genesuppression sequence, wherein transcription of the nucleic acid moleculein a host cell results in expression of a polypeptide encoded by thepolypeptide encoding sequence and suppression of a gene in the hostcell.

The present invention includes and provides a plant having a nucleicacid molecule comprising a first nucleic acid segment comprising apolypeptide encoding sequence and a second nucleic acid segmentcomprising a gene suppression sequence, wherein transcription of thenucleic acid molecule in a host cell results in expression of apolypeptide encoded by the polypeptide encoding sequence and suppressionof a gene in the host cell, wherein the first nucleic acid segment andthe second nucleic acid segment are operably linked to a single promotersequence.

The present invention also includes and provides a method ofsimultaneously altering the expression of more than one RNA moleculecomprising introducing into a plant cell a nucleic acid moleculecomprising a first nucleic acid segment comprising a polypeptideencoding sequence and a second nucleic acid segment comprising a genesuppression sequence, wherein transcription of the nucleic acid moleculein a host cell results in expression of a polypeptide encoded by thepolypeptide encoding sequence and suppression of a gene in the hostcell, wherein the first nucleic acid segment and the second nucleic acidsegment are operably linked to a single promoter sequence, and the firstnucleic acid segment and the second nucleic acid segment are expressed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of DNA construct elements in vector pMON75565.

FIG. 2 is a schematic of DNA construct elements in vector pMON75571.

FIGS. 3A and 3B are graphs depicting the percentage of alpha-tocopherolin the total tocopherol content of Arabidopsis seeds from the R₂generation of plants transformed with pMON75565 or pMON75571,respectively.

FIG. 4 is a graph representing the average seed oil and oleic fatty acid(18:1) levels in selected Arabidopsis seeds from the R₃ generation ofplants transformed with pMON75565.

FIGS. 5A and 5B are graphs depicting the total tocopherol levels (FIG.5A) and percentage of alpha-tocopherol in the total tocopherol content(FIG. 5B) of Arabidopsis seeds from the R₃ generation of plantstransformed with pMON75565.

DETAILED DESCRIPTION OF THE INVENTION

Description of Nucleic Acid Sequences

SEQ ID NO: 1 sets forth a nucleic acid sequence of a DNA molecule thatencodes a Gossypium hirsutum gamma-tocopherol methyltransferase.

SEQ ID NOs: 2 and 3 set forth nucleic acid sequences of primers for usein amplifying a Gossypium hirsutum gamma methyl transferase.

SEQ ID NO: 4 is the 1405 nucleotide long DNA sequence of the RNAioperative element found at bases 3345–4947 of pMON75565. SEQ ID NO:4comprises in 5′ to 3′ direction a sense-oriented 3′UTR sequence fromArabidopsis thaliana FAD2 (bases 1–135) linked to a sense-orientedintron sequence with splice sites removed from Arabidopsis thaliana FAD2(bases 144–1275) linked to an antisense oriented 3′UTR sequence fromArabidopsis thaliana FAD2 (bases 1281–1405). FAD2 intron elementsessentially as in SEQ ID NO:4 are found within pMON75565 at bases3687–4818 and within SEQ ID:5 at bases 3396–4515.

SEQ ID NO:5 is the 8179 nucleotide long DNA sequence of thetransformation insertion element between Agrobacterium tumefaciensborder elements from vector pMON75565, i.e. the elements of a firsttranscription unit for simultaneously increasing expression of GMT anddecreasing expression of Δ12 desaturase by RNAi and a secondtranscription unit for a BAR marker.

SEQ ID NO:6 is the 7713 nucleotide long DNA sequence of thetransformation insertion element between Agrobacterium tumefaciensborder elements from vector pMON75571, i.e. the elements of a firsttranscription units for simultaneously increasing expression of GMT anddecreasing expression of Δ12 desaturase by inton sense suppression and asecond transcription unit for a BAR marker.

Definitions:

As used herein, “gene” refers to a nucleic acid sequence thatencompasses a 5′ promoter region associated with the expression of thegene product, any intron and exon regions and 3′ untranslated regions(“UTR”) associated with the expression of the gene product.

As used herein, “a transgenic plant” is any plant that stablyincorporates a transgene in a manner that facilitates transmission ofthat transgene from a plant by any sexual or asexual method.

As used herein, “transgene” refers to a nucleic acid sequence associatedwith the expression of a gene introduced to a cell of an organism. Atransgene includes, but is not limited to, a gene endogenous to or agene not naturally occurring in the organism.

As used herein, “gene silencing” or “suppression” refers to thedown-regulation of gene expression by any method including, withoutlimitation, antisense suppression, sense suppression and sense intronsuppression. Such down-regulation can be a partial down-regulation.

As used herein, “a gene suppression sequence” is any nucleic acidsequence capable, when transcribed, of down-regulating gene expression.Such methods include but are not limited to antisense suppression, sensesuppression and sense intron suppression.

As used herein, “antisense suppression” refers to gene silencing that isinduced by the introduction of an antisense RNA molecule.

As used herein, “sense suppression” refers to gene silencing that isinduced by the introduction of a fragment of a gene in the senseorientation including, without limitation, a coding region or fragmentthereof.

As used herein, “sense intron suppression” refers to gene silencing thatis induced by the introduction of a intron in the sense orientation orfragment thereof of a gene. Sense intron suppression is described byFillatti in PCT WO 01/14538 A2, the entirety of which is incorporatedherein by reference.

When referring to proteins and nucleic acids herein, the use of plaincapitals, e.g., “GMT” or “FAD2,” indicates a reference to an enzyme,protein, polypeptide, or peptide, and the use of italicized capitals,e.g., “GMT” or “FAD2,” refers to nucleic acids, including withoutlimitation, genes, cDNAs, and mRNAs.

When referring to agents such as proteins and nucleic acids herein,“derived” refers to obtaining a protein or nucleic acid from a knownprotein or nucleic acid either directly (for example, by looking at thesequence of a known protein or nucleic acid and preparing a protein ornucleic acid having a sequence similar, at least in part, to thesequence of the known protein or nucleic acid) or indirectly (forexample, by obtaining a protein or nucleic acid from an organism whichis related to a known protein or nucleic acid). Other methods of“deriving” a protein or nucleic acid from a known protein or nucleicacid are known to one of skill in the art.

When referring to nucleic acid constructs herein, it is understood thatthe construct may be in linear or circular form.

As used herein, “a nucleic acid segment” is a portion of a largernucleic acid molecule. Such nucleic acid segments can, for example,without limitation, comprise a polypeptide encoding sequence or a genesuppression sequence or both.

As used herein, “RNAi,” and “dsRNA,” refer to gene silencing that isinduced by the introduction of a double-stranded RNA molecule.

As used herein, a “dsRNA molecule” and an “RNAi molecule” both refer toa double-stranded RNA molecule capable, when introduced into a cell ororganism, of at least partially reducing the level of an mRNA speciespresent in a cell or a cell of an organism.

As used herein, an “intron dsRNA molecule” and an “intron RNAi molecule”both refer to a double-stranded RNA molecule capable, when introducedinto a cell or organism, of at least partially reducing the level of anmRNA species present in one or more cells where the double-stranded RNAmolecule exhibits sufficient identity to an intron of a gene present inthe cell or organism to reduce the level of an mRNA containing thatintron sequence.

The term “non-coding” refers to sequences of nucleic acid molecules thatdo not encode part or all of an expressed protein. Non-coding sequencesinclude but are not limited to introns, promoter regions, 3′untranslated regions (“3′UTR”), and 5′ untranslated regions (“5′UTR”).

The term “intron” as used herein refers to the normal sense of the termas meaning a segment of a nucleic acid molecule, usually DNA, that doesnot encode part of or all of an expressed protein, and which, inendogenous conditions, is transcribed into RNA molecules, but which isspliced out of the endogenous RNA before the RNA is translated into aprotein.

The term “exon” as used herein refers to the normal sense of the term asmeaning a segment of nucleic acid molecules, usually DNA, which encodespart of or all of an expressed protein.

As used herein, a promoter that is “operably linked” to one or morenucleic acid sequences is capable of driving expression of one or morenucleic acid sequences, including multiple coding or non-coding nucleicacid sequences arranged in a polycistronic configuration or construct.

As used herein, a “plant promoter” includes, without limitation, a plantviral promoter and a synthetic, chimeric or hybrid promoter, which is asingle transcriptional unit, capable of functioning in a plant cell topromote the expression of an mRNA.

A “polycistronic configuration” or “polycistronic construct” is aconfiguration which comprises nucleic acid sequences of more than onegene. It is understood that within a “polycistronic configuration” theremay be sequences that correspond to exons, introns or both, and a“polycistronic configuration” might, for example without limitation,contain sequences that correspond to one or more exons from one gene andone or more introns from a second gene.

As used herein, a “polycistronic gene” or “polycistronic mRNA” is anygene or mRNA that contains nucleic acid sequences which correspond tonucleic acid sequences of more than one gene. It is understood that suchpolycistronic genes or mRNAs may contain sequences that correspond toexons, introns or both and that a recombinant polycistronic gene or mRNAmight, for example without limitation, contain sequences that correspondto one or more exons from one gene and one or more introns from a secondgene.

As used herein, “physically linked” nucleic acid sequences are nucleicacid sequences that are found on a single nucleic acid molecule.

As used herein, “expression” refers to the process of transcription andtranslation.

As used herein, “simultaneous expression” of more than one agent such asan mRNA or protein refers to the expression of an agent at the same timeas another agent. Such expression may only overlap in part and may alsooccur in different tissues or at different levels.

As used herein, “simultaneously altering expression” of more than oneagent such as an mRNA or protein refers to altering the expression of anagent at the same time as altering the expression of another agent. Suchexpression of the more than one agent may be altered in differenttissues or at different levels.

As used herein, “coexpression” of more than one agent such as an mRNA orprotein refers to the simultaneous expression of an agent at the sametime and in the same cell or tissue as another agent.

As used herein, “coordinated expression” of more than one agent” refersto the coexpression of more than one agent when the expression of suchagents is carried out utilizing a shared or identical promoter.

As used herein, an “at least partially enhanced” or an “increased” levelof an agent such as a protein or mRNA is at least partially enhanced orincreased if the level of that agent is increased relative to the levelthat that agent is present in a cell, tissue, plant or organism with asimilar genetic background but lacking an introduced nucleic acidmolecule encoding the protein or mRNA.

As used herein, a “polypeptide” comprises fifteen or greater amino acidresidues.

As used herein, a “peptide” contains 14 or fewer amino acid residues.

As used herein, an “enhanced” level of an agent such as a protein,polypeptide, peptide, or mRNA is enhanced if the level of that agent isincreased at least 25% relative to the level that that agent is presentin a cell, tissue, plant or organism with a similar genetic backgroundbut lacking an introduced nucleic acid molecule encoding the protein ormRNA.

As used herein, the level of an agent such as a protein, polypeptide,peptide, or mRNA is “substantially enhanced” if the level of that agentis increased at least 75% relative to the level that that agent ispresent in a cell, tissue, plant or organism with a similar geneticbackground but lacking an introduced nucleic acid molecule encoding theprotein or mRNA.

As used herein, “a reduction” of the level of an agent such as aprotein, polypeptide, peptide, or mRNA means that the level is decreasedrelative to a cell, tissue, plant or organism with a similar geneticbackground but lacking a nucleic acid sequence capable of reducing theagent.

As used herein, “at least a partial reduction” of the level of an agentsuch as a protein, polypeptide, peptide, or mRNA means that the level isdecreased at least 25% relative to a cell, tissue, plant or organismwith a similar genetic background but lacking a nucleic acid sequencecapable of reducing the agent.

As used herein, “a substantial reduction” of the level of an agent suchas a protein, polypeptide, peptide, or mRNA means that the level isdecreased relative to a cell, tissue, plant or organism with a similargenetic background but lacking a nucleic acid sequence capable ofreducing the agent, where the decrease in the level of the agent is atleast 75%.

As used herein, “an effective elimination” of an agent such as aprotein, polypeptide, peptide, or mRNA is relative to a cell, tissue,plant or organism with a similar genetic background but lacking anucleic acid sequence capable of decreasing the agent, where thedecrease in the level of the agent is greater than 95%.

As used herein, “heterologous” means not naturally occurring together.

As used herein, “an endogenous gene” is any gene that is not introducedinto a host by transformation or transfection.

As used herein, “total oil level” refers to the total aggregate amountof fatty acid without regard to the type of fatty acid.

As used herein, an “altered seed oil composition” refers to a seedcomposition in which the relative percentages of the types of fattyacids are altered.

As used herein, any range set forth is inclusive of the end points ofthe range unless otherwise stated.

Agents of the invention will preferably be “biologically active” withrespect to either a structural attribute, such as the capacity of anucleic acid molecule to hybridize to another nucleic acid molecule, orthe ability of a protein to be bound by an antibody (or to compete withanother molecule for such binding). Alternatively, such biologicalactivity may be catalytic and thus involve the capacity of the agent tomediate a chemical reaction or response.

Agents will preferably be “substantially purified.” The term“substantially purified,” as used herein, refers to a molecule separatedfrom substantially all other molecules normally associated with it inits native environmental conditions. More preferably a substantiallypurified molecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, greaterthan 75% free, preferably greater than 90% free, and most preferablygreater than 95% free from the other molecules (exclusive of solvent)present in the natural mixture. The term “substantially purified” is notintended to encompass molecules present in their native environmentalconditions.

Agents of the invention may also be recombinant. As used herein, theterm “recombinant” means any agent (e.g., including but not limited toDNA, RNA, peptide), that is, or results, however indirectly, from humanmanipulation of a nucleic acid molecule or peptide.

Agents of the invention may be labeled with reagents that facilitatedetection of the agent (e.g., fluorescent labels, Prober et al., Science238:336–340 (1987); Albarella et al., EP 144914; chemical labels,Sheldon et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.4,563,417; modified bases, Miyoshi et al., EP 119448).

As used herein, “% identity” is determined using the followingparameters and algorithm: Smith Waterman algorithm is used to determineidentity. Parameters for polypeptide sequence comparison typicallyinclude the following: Algorithm: Needleman and Wunsch, J. Mol. Biol.48:443–453 (1970). Comparison matrix: BLOSSUM62 from Hentikoff andHentikoff, Proc. Natl. Acad. Sci. USA 89:10915–10919 (1992). GapPenalty: 12; Gap Length Penalty: 4. A program that can be used withthese parameters is publicly available as the “gap” program fromGenetics Computer Group (“GCG”), Madison, Wis. The above parametersalong with no penalty for end gap are the default parameters for peptidecomparisons. Parameters for nucleic acid molecule sequence comparisonare the following: Algorithm: Needleman and Wunsch, J. Mol. Bio.48:443–453 (1970). Comparison matrix: matches—+10, mismatches=0; GapPenalty: 50; Gap Length Penalty: 3. “% identity” is determined using theabove parameters as the default parameters for nucleic acid moleculesequence comparisons and the “gap” program from GCG, version 10.2.

As used herein, a gamma-tocopherol methyltransferase (also referred toas GMT, γ-GMT, γ-MT, γ-TMT or gamma-methyltransferase) is anypolypeptide that is capable of specifically catalyzing the conversion ofγ-tocopherol into α-tocopherol. In certain plant species such assoybean, GMT can also catalyze the conversion of δ-tocopherol toβ-tocopherol. In other plants, GMT can also catalyze the conversion ofδ-tocotrienol to β-tocotrienol.

As used herein, a “FAD2”, “Δ12 desaturase” or “omega-6 desaturase” geneis a gene that encodes an enzyme capable of catalyzing the insertion ofa double bond into a fatty acyl moiety at the twelfth position countedfrom the carboxyl terminus.

Nucleic Acid Molecules, Constructs and Vectors

Vector systems suitable for introducing transforming DNA into a hostplant cell include, but are not limited to, binary bacterial artificialchromosome (BIBAC) vectors (Hamilton et al., Gene 200:107–116 (1997));RNA viral vectors (Della-Cioppa et al., Ann. N.Y. Acad. Sci. 792(Engineering Plants for Commercial Products and Applications):57–61(1996)); plant selectable YAC (Yeast Artificial Chromosome) vectors suchas those described in Mullen et al., Molecular Breeding 4:449–457(1988); cosmids; and bacterial artificial chromosomes (BACs), and suchvector systems can be utilized with nucleic acid molecules of thepresent invention. In one aspect of the invention such vectors contain anucleic acid molecule comprising a first nucleic acid segment comprisinga polypeptide encoding sequence and a second nucleic acid segmentcomprising a gene suppression sequence, wherein transcription of saidnucleic acid molecule in a host cell results in expression of apolypeptide encoded by the polypeptide encoding sequence and suppressionof a gene in the host cell. In one aspect, the first nucleic acid andthe second nucleic acid segment are operably linked to a single promotersequence. A second nucleic acid segment may be expressed, for example,without limitation, as a dsRNA molecule, an RNAi molecule, an introndsRNA molecule, or an intron RNAi molecule. In an aspect of the presentinvention, such first nucleic acid segment and second nucleic acidsegment can be expressed, coexpressed, or coordinately expressed in ahost cell and, upon expression, the RNA encoded by the second nucleicacid segment is suppressed.

A. Promoter

In an aspect of the present invention, nucleic acid molecules,constructs or vectors contain a promoter that is operably linked to oneor more nucleic acid sequences. Any promoter that functions in a plantcell to cause the production of an mRNA molecule, such as thosepromoters described herein, without limitation, can be used. In apreferred embodiment, the promoter is a plant promoter.

A number of promoters that are active in plant cells have been describedin the literature. These include, but are not limited to, the nopalinesynthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci. USA84:5745–5749 (1987)), the octopine synthase (OCS) promoter (which iscarried on tumor-inducing plasmids of Agrobacterium tumefaciens), thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al., Plant Mol. Biol. 9:315–324 (1987)), and theCaMV 35S promoter (Odell et al., Nature 313:810–812 (1985)), the figwortmosaic virus 35S-promoter (U.S. Pat. No. 5,378,619), the light-induciblepromoter from the small subunit of ribulose-1,5-bis-phosphatecarboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl.Acad. Sci. USA 84:6624–6628 (1987)), the sucrose synthase promoter (Yanget al., Proc. Natl. Acad. Sci. USA 87:4144–4148 (1990)), the R genecomplex promoter (Chandler et al., Plant Cell 1:1175–1183 (1989)) andthe chlorophyll a/b binding protein gene promoter. These promoters havebeen used to create DNA constructs that have been expressed in plants(See, e.g., PCT WO 84/02913). The CaMV 35S promoters are preferred foruse in plants. See also U.S. Pat. No. 6,437,217, which discloses a maizeRS81 promoter; U.S. Pat. No. 5,641,876, which discloses a rice actinpromoter; U.S. Pat. No. 6,426,446, which discloses a maize RS324promoter; U.S. Pat. No. 6,429,362, which discloses a maize PR-1promoter; U.S. Pat. No. 6,232,526, which discloses a maize A3 promoter;and U.S. Pat. No. 6,177,611, which discloses constitutive maizepromoter, all of which are incorporated by reference. The rice actin 1promoter with a rice actin intron is especially useful in the practiceof the present invention.

Particularly preferred promoters can also be used to express a nucleicacid molecule of the present invention in seeds or fruits. Indeed, in apreferred embodiment, the promoter used is a seed specific promoter.Examples of such promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)),phaseolin (Bustos et al., Plant Cell 1(9):839–853 (1989)), soybeantrypsin inhibitor (Riggs et al., Plant Cell 1(6):609–621 (1989)), ACP(Baerson et al., Plant Mol. Biol. 22(2):255–267 (1993)), stearoyl-ACPdesaturase (Slocombe et al., Plant Physiol. 104(4):167–176 (1994)),soybean a′ subunit of b-conglycinin (soy 7 s promoter, (Chen et al.,Proc. Natl. Acad. Sci. USA 83:8560–8564 (1986))), fatty acid elongation(FAE1) promoter (PCT WO 01/11061), and oleosin (see, for example, Honget al., Plant Mol. Biol. 34(3):549–555 (1997)). Further examples includethe promoter for β-conglycinin (Chen et al., Dev. Genet. 10: 112–122(1989)). Preferred promoters for expression in the seed are 7 S andnapin promoters.

Also included are the zein promoters, which are a group of storageproteins found in corn endosperm. Genomic clones for zein genes havebeen isolated (Pedersen et al., Cell 29:1015–1026 (1982); Russell etal., Transgenic Res. 6(2):157–168 (1997)) and the promoters from theseclones, including the 15 kD, 16 kD, 19 kD, 22 kD, and 27 kD genes, couldalso be used. Other promoters known to function, for example in corn,include the promoters for the following genes: waxy, Brittle, Shrunken2, Branching enzymes I and II, starch synthases, debranching enzymes,oleosins, glutelins and sucrose synthases. A particularly preferredpromoter for corn endosperm expression is the promoter for the glutelingene from rice, more particularly the Osgt-1 promoter (Zheng et al.,Mol. Cell Biol. 13:5829–5842 (1993)). Examples of promoters suitable forexpression in wheat include those promoters for the ADP glucosepyrosynthase (ADPGPP) subunits, the granule bound and other starchsynthase, the branching and debranching enzymes, theembryogenesis-abundant proteins, the gliadins and the glutenins.Examples of such promoters in rice include those promoters for theADPGPP subunits, the granule bound and other starch synthase, thebranching enzymes, the debranching enzymes, sucrose synthases and theglutelins. A particularly preferred promoter is the promoter for riceglutelin, Osgt-1. Examples of such promoters for barley include thosefor the ADPGPP subunits, the granule bound and other starch synthase,the branching enzymes, the debranching enzymes, sucrose synthases, thehordeins, the embryo globulins and the aleurone specific proteins.

Tissue-specific expression of a protein of the present invention is aparticularly preferred embodiment. The tissue-specific promoters thatcan be used include the chloroplast glutamine synthetase GS2 promoterfrom pea (Edwards et al., Proc. Natl. Acad. Sci. USA 87:3459–3463(1990)), the chloroplast fructose-1,6-biphosphatase (FBPase) promoterfrom wheat (Lloyd et al., Mol. Gen. Genet. 225:209–216 (1991)), thenuclear photosynthetic ST-LS1 promoter from potato (Stockhaus et al.,EMBO J 8:2445–2451 (1989)), the serine/threonine kinase (PAL) promoterand the glucoamylase (CHS) promoter from Arabidopsis thaliana. Alsoreported to be active in photosynthetically active tissues are theribulose-1, 5-bisphosphate carboxylase (rbcS) promoter from easternlarch (Larix laricina), the promoter for the cab gene, cab6, from pine(Yamamoto et al., Plant Cell Physiol. 35:773–778 (1994)), the promoterfor the Cab-1 gene from wheat (Fejes et al., Plant Mol. Biol. 15:921–932(1990)), the promoter for the CAB-1 gene from spinach (Lubberstedt etal., Plant Physiol. 104:997–1006 (1994)), the promoter for the cab1Rgene from rice (Luan et al., Plant Cell 4:971–981 (1992)), the pyruvate,orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al.,Proc. Natl. Acad. Sci. USA 90: 9586–9590 (1993)), the promoter for thetobacco Lhcb1*2 gene (Cerdan et al., Plant Mol. Biol. 33:245–255(1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter(Truernit et al., Planta. 196:564–570 (1995)) and the promoter for thethylakoid membrane proteins from spinach (psaD, psaF, psaE, PC, FNR,atpC, atpD, cab, rbcS). Other promoters for the chlorophyll a/b-bindingproteins may also be utilized in the invention, such as the promotersfor LhcB gene and PsbP gene from white mustard (Sinapis alba; Kretsch etal., Plant Mol. Biol. 28:219–229 (1995)).

A number of promoters for genes with tuber-specific or tuber-enhancedexpression are known and can be used, including the class I patatinpromoter (Bevan et al., EMBO J 8:1899–1906 (1986); Jefferson et al.,Plant Mol. Biol. 14:995–1006 (1990)), the promoter for the potato tuberADPGPP genes, both the large and small subunits, the sucrose synthasepromoter (Salanoubat and Belliard, Gene 60:47–56 (1987), Salanoubat andBelliard, Gene 84:181–185 (1989)), the promoter for the major tuberproteins including the 22 kd protein complexes and protease inhibitors(Hannapel, Plant Physiol. 101:703–704 (1993)), the promoter for thegranule-bound starch synthase gene (GBSS) (Visser et al., Plant Mol.Biol. 17:691–699 (1991)) and other class I and II patatins promoters(Koster-Topfer et al., Mol. Gen. Genet. 219:390–396 (1989); Mignery etal., Gene. 62:27–44 (1988)).

Root specific promoters may also be used. An example of such a promoteris the promoter for the acid chitinase gene (Samac et al., Plant Mol.Biol. 25:587–596 (1994)). Expression in root tissue could also beaccomplished by utilizing the root specific subdomains of the CaMV35Spromoter that have been identified (Lam et al., Proc. Natl. Acad. Sci.USA 86:7890–7894 (1989)). Other root cell specific promoters includethose reported by Conkling et al. (Conkling et al., Plant Physiol.93:1203–1211 (1990)).

The promoters used in the nucleic acid constructs of the presentinvention may be modified, if desired, to affect their controlcharacteristics. Promoters can be derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression. Such enhancers are known in theart. By including an enhancer sequence with such constructs, theexpression of the selected protein may be enhanced. These enhancersoften are found 5′ to the start of transcription in a promoter thatfunctions in eukaryotic cells, but can often be inserted in the forwardor reverse orientation 5′ or 3′ to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Deemed to beparticularly useful as enhancers are the 5′ introns of the rice actin 1and rice actin 2 genes. Examples of other enhancers which could be usedin accordance with the invention include elements from the CaMV 35Spromoter, octopine synthase genes, the maize alcohol dehydrogenase gene,the maize shrunken 1 gene and promoters from non-plant eukaryotes.

Where an enhancer is used in conjunction with a promoter for theexpression of a selected protein, it is believed that it will bepreferred to place the enhancer between the promoter and the start codonof the selected coding region. However, one also could use a differentarrangement of the enhancer relative to other sequences and stillrealize the beneficial properties conferred by the enhancer. Forexample, the enhancer could be placed 5′ of the promoter region, withinthe promoter region, within the coding sequence (including within anyother intron sequences which may be present), or 3′ of the codingregion.

In addition to introns with enhancing activity, other types of elementscan influence gene expression. For example, untranslated leadersequences predicted to enhance gene expression as well as “consensus”and preferred leader sequences have been identified. Preferred leadersequences are contemplated to include those which have sequencespredicted to direct optimum expression of the attached coding region,i.e., to include a preferred consensus leader sequence which mayincrease or maintain mRNA stability and prevent inappropriate initiationof translation. The choice of such sequences will be known to those ofskill in the art in light of the present disclosure. Sequences that arederived from genes that are highly expressed in plants, and in maize inparticular, will be most preferred. For example, sequences derived fromthe small subunit of ribulose bisphosphate carboxylase (RUBISCO).

In general it is preferred to introduce heterologous DNA randomly, i.e.at a non-specific location, in the genome. In special cases it may beuseful to target heterologous nucleic acid insertion in order to achievesite specific integration, e.g. to replace an existing gene in thegenome. In some other cases it may be useful to target a heterologousnucleic acid integration into the genome at a predetermined site fromwhich it is known that gene expression occurs. Several site specificrecombination systems exist which are known to function in plantsincluding cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT asdisclosed in U.S. Pat. No. 5,527,695.

Additional promoters that may be utilized are described, for example, inU.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858; 5,608,144;5,614,399; 5,633,441; 5,633,435; and 4,633,436. In addition, a tissuespecific enhancer may be used (Fromm et al., Plant Cell 1:977–984(1989)).

B. Nucleic Acid Molecules

In an aspect of the invention, the nucleic acid molecule comprises anucleic acid sequence, which when introduced into a cell or organism, iscapable of simultaneously overexpressing, expressing, coexpressing orcoordinately expressing one or more RNA molecules to produce one or moreproteins, fragments thereof, polypeptides, or peptides while expressingone or more other RNA molecules capable of suppressing the level of oneor more RNA molecules expressed in the cell or organism.

In this aspect of the present invention any protein, fragment thereof,polypeptide, or peptide can be expressed and any RNA molecule can besuppressed. Nucleic acid sequences encoding such proteins, fragmentsthereof, polypeptides, and peptides as well as nucleic acid sequencesuseful in the suppression of one or more mRNA molecules expressed in thecell or organism can be derived, for example, without limitation, from agene, fragment thereof, cDNA, fragment thereof, etc.

A gene of the present invention can be any gene, whether endogenous orintroduced. Nucleic acid sequences of such genes can be derived from amultitude of sources, including, without limitation, databases such asEMBL and Genbank found at www-ebi.ac.uk/swisprot/; www-expasy.ch/;www-embl.heidelberg.de/; and www-ncbi.nlm.nih.gov. Nucleic acidsequences of such genes can also be derived, without limitation, fromsources such as the GENSCAN program found athttp-genes.mit.edu/GENSCAN.html. In a further embodiment, additionalgenes may be obtained by any method by which additional genes may beidentified. In a preferred embodiment, an additional gene may beobtained by screening a genomic library with a probe of known genesequences. The gene may then be cloned and confirmed. Additional genesmay, for example without limitation, be amplified by polymerase chainreaction (PCR) and used in an embodiment of the present invention. Inaddition, other nucleic acid sequences of genes will be apparent to oneof ordinary skill in the art.

Any of a variety of methods may be used to obtain one or more genes.Automated nucleic acid synthesizers may be employed for this purpose,and to make a nucleic acid molecule that has a sequence also found in acell or organism. In lieu of such synthesis, nucleic acid molecules maybe used to define a pair of primers that can be used with the PCR toamplify and obtain any desired nucleic acid molecule or fragment of afirst gene.

In a preferred aspect, the gene, mRNA or protein is a non-viral gene,mRNA or protein. In another preferred aspect, the gene, RNA or proteinis an endogenous gene, RNA or protein. In a preferred aspect, a gene isa GMT gene. A preferred GMT gene of the present invention is a plant orcyanobacterial GMT, more preferably a GMT that is also found in anorganism selected from the group consisting of Arabidopsis, rice, corn,cotton, cuphea, oilseed rape, tomato, soybean, marigold, sorghum, andleek, most preferably a GMT that is also found in an organism selectedfrom the group consisting of Arabidopsis thaliana, Oryza sativa, Zeamays, Gossypium hirsutum, Cuphea pulcherrima, Brassica napus,Lycopersicon esculentum, Glycine max, Tagetes erecta, and Liliumasiatic. Representative sequences for GMT genes include, withoutlimitation, those set forth in U.S. patent application Ser. No.10/219,810, filed on Aug. 16, 2002.

In an aspect, another preferred gene of the present invention is a FAD2gene. Representative sequences for FAD2 include, without limitation,those set forth in U.S. application Ser. No. 10/176,149, filed Jun. 21,2002, and U.S. patent application Ser. No. 09/638,508, filed Aug. 11,2000, and U.S. Provisional Application Ser. No. 60/151,224, filed Aug.26, 1999, and U.S. Provisional Application Ser. No. 60/172,128, filedDec. 17, 1999. In a preferred aspect a GMT protein is expressed and theexpression of a FAD2 protein is suppressed.

In an aspect of the present invention, a nucleic acid moleculecomprising a first nucleic acid segment comprising a polypeptideencoding sequence and a second nucleic acid segment comprising a genesuppression sequence, wherein transcription of the nucleic acid moleculein a host cell results in expression of a polypeptide encoded by thepolypeptide encoding sequence and suppression of a gene in said hostcell, where the first nucleic acid segment and the second nucleic acidsegment are operably linked to a single promoter sequence.

In a preferred aspect of the present invention the nucleic acid moleculefurther comprises nucleotide sequences encoding a plastid transitpeptide operably fused to a nucleic acid molecule of the presentinvention that encodes a protein, fragment thereof, polypeptide, orpeptide.

A nucleic acid molecule or protein, fragment thereof, polypeptide, orpeptide of the present invention may differ in either nucleic acid oramino acid sequence from a gene or its translated product butnonetheless share a percentage identity with a nucleic acid or aminoacid sequence from a gene. “Identity,” as is well understood in the art,is a relationship between two or more polypeptide sequences or two ormore nucleic acid molecule sequences, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness between polypeptide or nucleic acid molecule sequences, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods.

In another aspect, the nucleic acid sequence of the nucleic acidmolecules of the present invention can comprise sequences that differfrom those encoding a protein, fragment thereof, polypeptide, or peptidedue to the fact that a protein, fragment thereof, polypeptide, orpeptide can have one or more conservative amino acid changes, andnucleic acid sequences coding for the polypeptide can therefore havesequence differences.

It is well known in the art that one or more amino acids in a nativesequence can be substituted with other amino acid(s), the charge andpolarity of which are similar to that of the native amino acid, i.e., aconservative amino acid substitution. Hydropathic index of amino acidsmay also be considered when making amino acid changes. The importance ofthe hydropathic amino acid index in conferring interactive biologicalfunction on a protein is generally understood in the art (Kyte andDoolittle, J. Mol. Biol. 157:105–132 (1982)). It is also understood inthe art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein. In making such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

Due to the degeneracy of the genetic code, different nucleotide codonsmay be used to code for a particular amino acid. A host cell oftendisplays a preferred pattern of codon usage. Structural nucleic acidsequences are preferably constructed to utilize the codon usage patternof the particular host cell. This generally enhances the expression ofthe structural nucleic acid sequence in a transformed host cell. Any ofthe above-described nucleic acid and amino acid sequences may bemodified to reflect the preferred codon usage of a host cell or organismin which they are contained. Modification of a structural nucleic acidsequence for optimal codon usage in plants is described in U.S. Pat. No.5,689,052.

Preferred embodiments of the invention include nucleic acid moleculesthat comprise a first, second or both nucleic acid segment(s), which isat least 50%, 60%, or 70% identical over its entire length to a plantgene. More preferable are first, second or both nucleic acid segmentswhich comprise a region that is at least 80% or at least 85% identicalover its entire length to a plant gene. In this regard first and secondnucleic acid segments at least 90% identical over their entire lengthare particularly preferred, those at least 95% identical are especiallypreferred. Further, those with at least 97% identity are highlypreferred and those with at least 98% and at least 99% identity areparticularly highly preferred, with those exhibiting 100% identity beingthe most highly preferred.

A subset of the first or second nucleic acid segment of the nucleic acidmolecules of the invention includes fragment nucleic acid molecules.Fragment nucleic acid molecules may consist of significant portion(s)of, or indeed most of, a plant gene. Alternatively, fragments maycomprise smaller oligonucleotides, having from about 15 to about 400contiguous nucleotide residues and more preferably, about 15 to about 45contiguous nucleotide residues, about 20 to about 45 contiguousnucleotide residues, about 15 to about 30 contiguous nucleotideresidues, about 21 to about 30 contiguous nucleotide residues, about 21to about 25 contiguous nucleotide residues, about 21 to about 24contiguous nucleotide residues, about 19 to about 25 contiguousnucleotide residues, or about 21 contiguous nucleotides. In a preferredembodiment, a fragment shows 100% identity to a region of a plant gene.In another preferred embodiment, a fragment comprises a portion of alarger nucleic acid sequence. In another aspect, a fragment nucleic acidmolecule has a nucleic acid sequence that has at least 15, 25, 50, or100 contiguous nucleotides of a nucleic acid molecule of the presentinvention. In a preferred embodiment, a nucleic acid molecule has anucleic acid sequence that has at least 15, 25, 50, or 100 contiguousnucleotides of a plant gene.

It is understood that a nucleic acid of the present invention can be ineither orientation and that such molecules can be in a sense orantisense orientation.

A first nucleic acid segment can be physically linked to or part of apolycistronic construct with a second nucleic acid segment. Nucleic acidsequences within a first or second nucleic acid segment can bephysically linked to or part of a polycistronic construct with othernucleic acid segments. A promoter can be physically linked to or part ofa polycistronic construct with a first nucleic segment and secondnucleic acid segment. Such polycistronic constructs can be capable ofexpressing a polycistronic mRNA.

i. First Nucleic Acid Segment Capable of Being Transcribed as One orMore RNAs

A first nucleic acid segment can be any nucleic acid sequence that iscapable of being transcribed and expressed as an mRNA. In an aspect, thenucleic acid sequence corresponds to a nucleic acid sequence that isalso found in a naturally occurring gene or part of a gene such as atranscribed segment of a gene. Such a gene can be any gene from anyorganism. In a preferred aspect the gene is from a plant. In anotherpreferred aspect the gene is from a microorganism. An illustrative geneis a GMT gene. A first nucleic acid segment which is transcribed andexpressed as an mRNA can be translated into a protein, fragment thereof,polypeptide, or peptide. In one aspect the proteins, fragments thereof,polypeptides, or peptides are also endogenous to the host. In anotheraspect the proteins, fragments thereof, polypeptides, or peptides arenot normally found in the plant. In a further aspect the amino acidsequence of the proteins, fragments thereof, polypeptides, or peptidesare not found in a non-transformed host.

It is also understood that a first nucleic acid segment can containsequences that encode for more than one protein, fragment thereof,polypeptide, or peptide. In this aspect, the proteins, fragmentsthereof, polypeptides, or peptides may be a combination of proteins,fragments thereof, polypeptides, or peptides endogenous to the host, notnormally found in the plant, or not found in a non-transformed host. Inthis aspect, a first nucleic acid segment can encode for two, three,four, five, or more than five proteins, fragments thereof, polypeptides,or peptides.

ii. Second Nucleic Acid Sequence Capable of Suppressing One or More RNAs

A second nucleic acid segment can be any nucleic acid sequence which,when introduced into a cell or organism, is capable of effectivelyeliminating, substantially reducing, at least partially reducing orreducing the level of an mRNA transcript or protein encoded by a gene.In an aspect of the present invention, a gene is an endogenous gene. Inan aspect of the present invention, a gene is a plant gene. Anillustrative gene is a FAD2 gene.

It is also understood that a second nucleic acid segment can be anynucleic acid sequence, which, when introduced into a cell or organism,is capable of effectively eliminating, substantially reducing, at leastpartially reducing or reducing the level of one, two, three, four, five,or more mRNAs. It also understood in this aspect that an individual mRNAmay be suppressed by different methodologies, for example RNAi andantisense suppression.

In an aspect of the invention, the second nucleic acid sequence of thepresent invention, which is preferably a dsRNA construct, preferably asense RNA construct, or preferably an antisense RNA construct, iscapable of providing at least a partial reduction, more preferably asubstantial reduction, or most preferably effective elimination ofanother agent such as a protein or mRNA. In an aspect of the presentinvention, the other agent is a FAD2 protein or mRNA encoded by a FAD2gene.

In another aspect, the level of one or more agents is reduced, at leastpartially reduced, substantially reduced or effectively eliminated whilethe level of one or more simultaneously, co-expressed or coordinatelyexpressed agents is at least partially enhanced, at least enhanced, orsubstantially enhanced.

In a further embodiment, a nucleic acid molecule, when introduced into acell or organism, selectively increases the level of a first protein orRNA transcript or both encoded by a first gene and at the same timereduces the level of a second protein, transcript or both encoded by asecond gene, and also alters the alpha-tocopherol content, the oilcomposition, and the oil level of the cell or organism.

Multiple methodologies can be used to effectively eliminate,substantially reduce, or at least partially reduce the level of an mRNAtranscript or protein encoded by a gene. Such methods can result in genespecific silencing or in the silencing of multiple genes. Examples ofsuch gene silencing include, without limitation, those induced by theintroduction of a double-stranded RNA molecule, antisense, and senseRNA.

In another aspect, a second nucleic acid segment can be any nucleic acidsequence which, when introduced into a cell or organism, is capable ofeffectively eliminating, substantially reducing, at least partiallyreducing or reducing the level of two, three, four, five, or more thanfive mRNA transcripts or proteins encoded by a gene.

a. dsRNA

Double-stranded molecules which can be used for gene silencing includedsRNA molecules that comprise nucleic acid sequences corresponding to anucleic acid sequence found in a transcript. Such nucleic acid sequencesinclude, without limitation, nucleic acid sequences that encode for aprotein, fragment thereof, polypeptide, or peptide, and those thatcorrespond to transcribed introns, transcribed 3′ untranslated regions(UTRs), and transcribed 5′ UTRs.

One subset of the second nucleic acid sequence of the nucleic acidmolecules of the invention is a nucleic acid sequence which is expressedas a double-stranded RNA which comprises (1) a first RNA fragment thatexhibits identity to a transcribed region of a second gene which is tobe suppressed, and (2) a second RNA capable of forming a double-strandedRNA molecule with the first RNA. The first RNA fragment may consist ofsignificant portion(s) of, or indeed most of, a plant gene which is tobe suppressed.

In an aspect, a nucleic acid molecule of the present invention comprisesa nucleic acid sequence which exhibits sufficient homology to one ormore plant introns from a second plant gene, which when introduced intoa plant cell or plant as a dsRNA construct, is capable of effectivelyeliminating, substantially reducing, or at least partially reducing thelevel of an mRNA transcript or protein encoded by the gene from whichthe intron(s) was derived.

In an aspect, a nucleic acid molecule of the present invention comprisesa nucleic acid sequence which exhibits sufficient homology to one ormore plant exons from a second plant gene, which when introduced into aplant cell or plant as a dsRNA construct, is capable of effectivelyeliminating, substantially reducing, or at least partially reducing thelevel of an mRNA transcript or protein encoded by the gene from whichthe exon(s) was derived.

In an aspect, a nucleic acid molecule of the present invention comprisesa nucleic acid sequence which exhibits sufficient homology to one ormore plant transcribed 3′ UTR(s) from a second plant gene, which whenintroduced into a plant cell or plant as a dsRNA construct, is capableof effectively eliminating, substantially reducing, or at leastpartially reducing the level of an mRNA transcript or protein encoded bythe gene from which the 3′ UTR(s) was derived.

In an aspect, a nucleic acid molecule of the present invention comprisesa nucleic acid sequence which exhibits sufficient homology to one ormore plant transcribed 5′ UTR(s) from a second plant gene, which whenintroduced into a plant cell or plant as a dsRNA construct, is capableof effectively eliminating, substantially reducing, or at leastpartially reducing the level of an mRNA transcript or protein encoded bythe gene from which the 5′ UTR(s) was derived.

In another preferred aspect, a dsRNA construct contains exon sequences,but the exon sequences do not correspond to a sufficient part of a plantexon to be capable of effectively eliminating, substantially reducing,or at least partially reducing the level of an mRNA transcript orprotein encoded by a second gene from which the exon was derived.Strategies of suppressing gene expression with dsRNA constructs includethat set forth in U.S. Provisional Patent Application Ser. No.60/390,186, filed on Jun. 9, 2000.

b. Antisense Suppression

Antisense molecules which can be used for gene silencing include anymolecules that comprise nucleic acid sequences corresponding to acomplement of a nucleic acid sequence found in a transcript or partthereof or molecules with sufficient complementarity to act as antisensemolecules. Such molecules include sequences, without limitation, thatare the complement of those that encode for a protein, fragment thereofor polypeptide, and are the complement of those that correspond totranscribed introns, transcribed 3′ untranslated regions (UTRs), andtranscribed 5′ UTRs.

Antisense approaches are a way of preventing or reducing gene functionby targeting the genetic material (Mol et al., FEBS Lett. 268:427–430(1990)). The objective of the antisense approach is to use a sequencecomplementary to the target gene to block its expression and create amutant cell line or organism in which the level of a single chosenprotein is selectively reduced or abolished. The site of inactivationand its developmental effect can be manipulated by the choice ofpromoter for antisense genes or by the timing of external application ormicroinjection. Antisense can manipulate its specificity by selectingeither unique regions of the target gene or regions where it shareshomology to other related genes (Hiatt et al., In: Genetic Engineering,Setlow (ed.), Vol. 11, New York: Plenum 49–63 (1989)).

Antisense RNA techniques involve introduction of RNA that iscomplementary to the target mRNA into cells, which results in specificRNA:RNA duplexes being formed by base pairing between the antisensesubstrate and the target mRNA (Green et al., Annu. Rev. Biochem.55:569–597 (1986)). Under one embodiment, the process involves theintroduction and expression of an antisense gene sequence. Such asequence is one in which part or all of the normal gene sequences areplaced under a promoter in inverted orientation so that the ‘wrong’ orcomplementary strand is transcribed into a noncoding antisense RNA thathybridizes with the target mRNA and interferes with its expression(Takayama and Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155–184 (1990)).An antisense vector is constructed by standard procedures and introducedinto cells by transformation, transfection, electroporation,microinjection, infection, etc. The type of transformation and choice ofvector will determine whether expression is transient or stable. Thepromoter used for the antisense gene may influence the level, timing,tissue, specificity, or inducibility of the antisense inhibition.

c. Cosuppression or Sense Suppression

Sense suppression molecules which can be used for gene silencing includeany molecules that comprise nucleic acid sequences corresponding to anucleic acid sequence found in a transcript or part thereof or moleculeswith sufficient complementarity to act as sense molecules. Suchmolecules include sequences, without limitation, that encode for aprotein, fragment thereof or polypeptide, and those that correspond totranscribed introns, transcribed 3′ untranslated regions (UTRs), andtranscribed 5′ UTRs

Cosuppression is the reduction in expression levels, usually at thelevel of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene (Napoli et al., Plant Cell 2:279–289 (1990); van derKrol et al., Plant Cell 2:291–299 (1990)). Cosuppression may result fromstable transformation with a single copy nucleic acid molecule that ishomologous to a nucleic acid sequence found within the cell (Prolls andMeyer, Plant J. 2:465–475 (1992)) or with multiple copies of a nucleicacid molecule that is homologous to a nucleic acid sequence found withinthe cell (Mittlesten et al., Mol. Gen. Genet. 244:325–330 (1994)).Genes, even though different, linked to homologous promoters may resultin the cosuppression of the linked genes (Vaucheret, C. R. Acad. Sci.III316:1471–1483 (1993); Flavell, Proc. Natl. Acad. Sci. (U.S.A.)91:3490–3496 (1994); van Blokland et al., Plant J. 6:861–877 (1994);Jorgensen, Trends Biotechnol. 8:340–344 (1990); Meins and Kunz, In: GeneInactivation and Homologous Recombination in Plants, Paszkowski (ed.),pp. 335–348, Kluwer Academic, Netherlands (1994)).

iii. Suppression or Expression Nucleic Acid Molecules

In one aspect of the present invention, the present invention provides anucleic acid molecule which can encode for two, three, four, five, ormore than five proteins, fragments thereof, polypeptides, or peptidesoperably linked to a single promoter sequence.

In another aspect of the present invention, the present inventionprovides a nucleic acid molecule which, when introduced into a cell ororganism, is capable of effectively eliminating, substantially reducing,at least partially reducing or reducing the level of two, three, four,five, or more than five mRNA transcripts or proteins encoded by a gene,operably linked to a single promoter sequence.

C. Other Components of Construct/Vector

Constructs or vectors may also include, within the region of interest, anucleic acid sequence that acts, in whole or in part, to terminatetranscription of that region. A number of such sequences have beenisolated, including the Tr7 3′ sequence and the NOS 3′ sequence(Ingelbrecht et al., Plant Cell 1:671–680 (1989); Bevan et al., NucleicAcids Res. 11:369–385 (1983)). Regulatory transcript termination regionscan be provided in plant expression constructs of the present inventionas well. Transcript termination regions can be provided by the DNAsequence encoding the gene of interest or a convenient transcriptiontermination region derived from a different gene source, for example,the transcript termination region that is naturally associated with thetranscript initiation region. The skilled artisan will recognize thatany convenient transcript termination region that is capable ofterminating transcription in a plant cell can be employed in theconstructs of the present invention.

A vector or construct may also include regulatory elements. Examples ofsuch include the Adh intron 1 (Callis et al., Genes and Develop.1:1183–1200 (1987)), the sucrose synthase intron (Vasil et al., PlantPhysiol. 91:1575–1579 (1989)) and the TMV omega element (Gallie et al.,Plant Cell 1:301–311 (1989)). These and other regulatory elements may beincluded when appropriate.

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to: a neo gene (Potrykus et al., Mol. Gen. Genet.199:183–188 (1985)), which codes for kanamycin resistance and can beselected for using kanamycin, RptII, G418, hpt; a bar gene which codesfor bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al.,Bio/Technology 6:915–922 (1988); Reynaerts et al., Selectable andScreenable Markers, In Gelvin and Schilperoort, Plant Molecular BiologyManual, Kluwer, Dordrecht (1988)); aadA (Scofield et al., Mol. Gen.Genet. 244(2):189–96 (1994)), which encodes glyphosate resistance; anitrilase gene which confers resistance to bromoxynil (Stalker et al.,J. Biol. Chem. 263:6310–6314 (1988)); a mutant acetolactate synthasegene (ALS) which confers imidazolinone or sulphonylurea resistance(European Patent Application 154,204 (Sep. 11, 1985)); ALS (D'Halluin etal., Bio/Technology 10: 309–314 (1992)); and a methotrexate resistantDHFR gene (Thillet et al., J. Biol. Chem. 263:12500–12508 (1988)).

A vector or construct may also include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude: a β-glucuronidase or uidA gene (GUS) which encodes an enzymefor which various chromogenic substrates are known (Jefferson, PlantMol. Biol, Rep. 5:387–405 (1987); Jefferson et al., EMBO J. 6:3901–3907(1987)); an R-locus gene, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues(Dellaporta et al., Stadler Symposium 11:263–282 (1988)); a β-lactamasegene (Sutcliffe et al., Proc. Natl. Acad. Sci. USA 75:3737–3741 (1978)),a gene which encodes an enzyme for which various chromogenic substratesare known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene(Ow et al., Science 234:856–859 (1986)); a xylE gene (Zukowsky et al.,Proc. Natl. Acad. Sci. USA 80:1101–1105 (1983)) which encodes a catecholdioxygenase that can convert chromogenic catechols; an α-amylase gene(Ikatu et al., Bio/Technology 8:241–242 (1990)); a tyrosinase gene (Katzet al., J. Gen. Microbiol. 129:2703–2714 (1983)) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to melanin; and an α-galactosidase gene, which encodes anenzyme which will turn a chromogenic α-galactose substrate.

Included within the terms “selectable or screenable marker genes” arealso genes that encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes that canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins that are detectable,(e.g., by ELISA), small active enzymes that are detectable inextracellular solution (e.g., α-amylase, β-lactamase, phosphinothricintransferase), or proteins that are inserted or trapped in the cell wall(such as proteins that include a leader sequence such as that found inthe expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

Transgenic Plants, Parts Thereof and Plant Cells

Exogenous genetic material may be transferred into a plant cell and theplant cell can be regenerated into a whole, fertile or sterile plant orplant part. Exogenous genetic material is any genetic material, whethernaturally occurring or otherwise, from any source that is capable ofbeing inserted into any organism. Such exogenous genetic materialincludes, without limitation, nucleic acid molecules and constructs thatcomprise a nucleic acid sequence of the present invention, as set forthwithin.

In a preferred aspect, a plant cell or plant of the present inventionincludes a nucleic acid molecule comprising a first and second nucleicacid sequence, where the first nucleic acid sequence which, when it isexpressed, is capable of at least partially enhancing, increasing,enhancing, or substantially enhancing the level of an mRNA transcript orprotein and where the second nucleic acid sequence exhibits sufficienthomology to one or more plant genes such that when it is expressed, itis capable of effectively eliminating, substantially reducing, or atleast partially reducing the level of an mRNA transcript or proteinencoded by the gene from which it was derived or any gene which hashomology to that gene.

It is understood that any methodology that will suppress the expressionof a gene can be used.

In an aspect of the present invention, a plant cell or plant of thepresent invention includes a nucleic acid molecule that comprises anucleic acid sequence which is capable of increasing the protein,transcript or both encoded by a GMT gene and at the same timeselectively reducing the protein, transcript or both encoded by a FAD2gene.

In a preferred aspect, a plant cell or plant of the present inventionincludes a nucleic acid molecule that comprises a first nucleic acidsegment and a second nucleic acid segment, where the first nucleic acidsegment, the second nucleic acid segment, or both, are capable ofaltering seed oil composition. In a more preferred aspect, the firstnucleic acid sequence, when it is expressed, is capable of increasingthe level of alpha-tocopherol, and the second nucleic acid segmentexhibits sufficient homology to complements of one or more plant genessuch that when it is expressed, it is capable of increasing the level ofoleic acid or oil content, or both, the first nucleic acid sequence andthe second nucleic acid sequence being operably linked to a singlepromoter sequence.

Genetic material may be introduced into any species, for example,without limitation monocotyledons or dicotyledons, including, but notlimited to alfalfa, apple, Arabidopsis, banana, barley, Brassicacampestris, canola, castor bean, chrysanthemum, coffee, cotton,cottonseed, corn, crambe, cranberry, cucumber, dendrobium, dioscorea,eucalyptus, fescue, flax, gladiolus, liliacea, linseed, millet,muskmelon, mustard, oat, oil palm, oilseed rape, papaya, peanut,perennial, Phaseolus, potato, rapeseed, rice, rye, ryegrass, safflower,sesame, sorghum, soybean, sugarbeet, sugarcane, sunflower, tobacco,tomato, turfgrass, and wheat (Christou, INO: Particle Bombardment forGenetic Engineering of Plants, Biotechnology Intelligence Unit. AcademicPress, San Diego, Calif. (1996)), with alfalfa, Arabidopsis, Brassicacampestris, canola, castor bean, corn, cotton, cottonseed, crambe, flax,linseed, mustard, oil palm, oilseed rape, peanut, potato, rapeseed,safflower, sesame, soybean, sunflower, tobacco, tomato, and wheatpreferred, and Brassica campestris, canola, corn, oil palm, oilseedrape, peanut, rapeseed, safflower, soybean, and sunflower morepreferred. In a more preferred aspect, genetic material is transferredinto canola. In another more preferred aspect, genetic material istransferred into oilseed rape. In another particularly preferredembodiment, genetic material is transferred into soybean or corn.

Genetic material may also be introduced into a suitable cell such as aplant cell. The cell may be present in a multi-cellular environment. Inan aspect of the present invention, the multicellular environment may bein a transformed plant.

Genetic material may also be introduced into a cell or organism such asa mammalian cell, mammal, fish cell, fish, bird cell, bird, algae cell,algae, fungal cell, fungi, or bacterial cell. Preferred host andtransformants include: fungal cells such as Aspergillus, yeasts,mammals, particularly bovine and porcine, insects, bacteria, and algae.Particularly preferred bacteria are Agrobacteruim tumefaciens and E.coli.

The levels of products such as transcripts or proteins may be increasedor decreased or both throughout an organism such as a plant or localizedin one or more specific organs or tissues of the organism. For examplethe levels of products may be increased or decreased in one or more ofthe tissues and organs of a plant including without limitation: roots,tubers, stems, leaves, stalks, fruit, berries, nuts, bark, pods, seedsand flowers. A preferred organ is a seed.

In an aspect of the invention, after transformation of a plant or otherorganism with a nucleic acid of the present invention, the level of oneor more agents is at least partially enhanced, increased, enhanced, orsubstantially enhanced, while a second agent is simultaneouslyexpressed, coexpressed, or coordinately expressed with the first agent.

In another aspect, after transformation of a plant or other organismwith a nucleic acid of the present invention, the level of one or moreagents is at least partially enhanced, increased, enhanced, orsubstantially enhanced, while a second agent is simultaneouslyexpressed, coexpressed, or coordinately expressed, and the simultaneousexpression, coexpression or coordinate expression of the second agentresults in a reduction, preferably at least a partial reduction,substantial reduction or effective elimination of another agent.

In another aspect, after transformation of a plant or other organismwith a nucleic acid of the present invention, the level of one or moreagents is at least partially enhanced, increased, enhanced, orsubstantially enhanced, while a second agent is simultaneouslyexpressed, coexpressed, or coordinately expressed with two or greaterthan two agents.

In another aspect, after transformation of a plant or other organismwith a nucleic acid of the present invention, the level of one or moreagents is at least partially enhanced, increased, enhanced, orsubstantially enhanced, while a second agent is simultaneouslyexpressed, coexpressed, or coordinately expressed with three or greaterthan three agents.

In another aspect, after transformation of a plant or other organismwith a nucleic acid of the present invention, the level of one or moreagents is at least partially enhanced, increased, or substantiallyenhanced while additional agents are simultaneously expressed,coexpressed or coordinately expressed with the first agent and thesimultaneous expression, coexpression or coordinated expression of theadditional agents, preferably two or more, three or more, four or more,or five or more agents, result in at least partial reduction,substantial reduction or an effective elimination of more than oneagent, preferably two or more, three or more, four or more, or five ormore agents.

In an aspect, after transformation of a plant or other organism with anucleic acid of the present invention, one or more agents is at leastpartially enhanced, increased, enhanced, or substantially enhanced whileanother agent or agents is simultaneously expressed, coexpressed, orcoordinately expressed and such expression results in at least a partialreduction, a substantial reduction, or effective elimination of an agentor agents.

When levels of an agent are compared, such a comparison is preferablycarried out between organisms with a similar genetic background. In apreferred aspect, a similar genetic background is a background where theorganisms being compared share 50% or greater of their nuclear geneticmaterial. In a more preferred aspect a similar genetic background is abackground where the organisms being compared share 75% or greater, evenmore preferably 90% or greater of their nuclear genetic material. Inanother even more preferable aspect, a similar genetic background is abackground where the organisms being compared are plants, and the plantsare isogenic except for any genetic material originally introduced usingplant transformation techniques.

In a preferred aspect, the capability of a nucleic acid sequence topartially enhance, enhance or substantially enhance the level of anagent is carried out by a comparison of levels of mRNA transcripts. In apreferred aspect, the capability of a nucleic acid sequence to partiallyenhance, enhance, or substantially enhance the level of a gene relativeto another gene is carried out by a comparison of levels of proteins,fragments thereof or polypeptides encoded by the genes. In a preferredaspect, the capability of a nucleic acid sequence to reduce the level ofa gene relative to another gene is carried out by a comparison of levelsof mRNA transcripts. In a preferred aspect, the capability of a nucleicacid sequence to reduce the level of a gene relative to another gene iscarried out by a comparison of levels of proteins, fragments thereof orpolypeptides encoded by the genes. As used herein, mRNA transcriptsinclude processed and non-processed mRNA transcripts. As used herein,proteins, fragments thereof or polypeptides include proteins, fragmentsthereof or polypeptides with or without any post-translationalmodification. In another preferred aspect, the capability of a nucleicacid molecule to increase the level of a gene relative to another geneis carried out by a comparison of phenotype. In a preferred aspect, thecomparison of phenotype is a comparison of alpha-tocopherol content. Ina preferred aspect, the comparison of phenotype is a comparison of fattyacid composition. In a preferred aspect, the comparison of phenotype isa comparison of total oil level.

Methods of Introducing Nucleic Acid Molecules into Plants or Organisms

There are many methods for introducing nucleic acid molecules into plantcells. Suitable methods are believed to include virtually any method bywhich nucleic acid molecules may be introduced into a cell, such as byAgrobacterium infection or direct delivery of nucleic acid moleculessuch as, for example, by transfection, injection, projection,PEG-mediated transformation, by electroporation or by acceleration ofDNA coated particles, and the like. (Potrykus, Ann. Rev. Plant Physiol.Plant Mol. Biol. 42:205–225 (1991); Vasil, Plant Mol. Biol. 25:925–937(1994)). For example, electroporation has been used to transform cornprotoplasts (Fromm et al., Nature 312:791–793 (1986)).

Nucleic acids can also be introduced into an organism via methodsincluding, but not limited to, conjugation, endocytosis, andphagocytosis. Furthermore, the nucleic acid can be introduced into acell or organism derived from a plant, plant cell, algae, algae cell,fungus, fungal cell, bacterial cell, mammalian cell, fish cell, or birdcell. Particularly preferred microorganisms are E. coli andAgrobacterium species.

Technology for introduction of DNA into cells is well known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb,Virology 54:536–539 (1973)); (2) physical methods such as microinjection(Capecchi, Cell 22:479–488 (1980)), electroporation (Wong and Neumann,Biochem. Biophys. Res. Commun. 107:584–587 (1982); Fromm et al., Proc.Natl. Acad. Sci. USA 82:5824–5828 (1985); U.S. Pat. No. 5,384,253); thegene gun (Johnston and Tang, Methods Cell Biol. 43:353–365 (1994)); andvacuum infiltration (Bechtold et al., C.R. Acad. Sci. Paris, Life Sci.316:1194–1199. (1993)); (3) viral vectors (Clapp, Clin. Perinatol.20:155–168 (1993); Lu et al., J. Exp. Med. 178:2089–2096 (1993); Eglitisand Anderson, Biotechniques 6:608–614 (1988)); and (4) receptor-mediatedmechanisms (Curiel et al., Hum. Gen. Ther. 3:147–154 (1992); Wagner etal., Proc. Natl. Acad. Sci. USA 89:6099–6103 (1992)).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules into plant cells ismicroprojectile bombardment. This method has been reviewed by Yang andChristou (eds.), Particle Bombardment Technology for Gene Transfer,Oxford Press, Oxford, England (1994). Non-biological particles(microprojectiles) may be coated with nucleic acid molecules anddelivered into cells by a propelling force. Exemplary particles includethose comprised of tungsten, gold, platinum and the like.

A particular advantage of microprojectile bombardment, in addition to itbeing an effective way of reproducibly transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., Plant Physiol.87:671–674 (1988)) nor the susceptibility to Agrobacterium infection isrequired. An illustrative embodiment of a method for delivering DNA intocorn cells by acceleration is a biolistics α-particle delivery system,which can be used to propel particles coated with DNA through a screen,such as a stainless steel or Nytex screen, onto a filter surface coveredwith corn cells cultured in suspension. Gordon-Kamm et al., describesthe basic procedure for coating tungsten particles with DNA (Gordon-Kammet al., Plant Cell 2:603–618 (1990)). The screen disperses the tungstennucleic acid particles so that they are not delivered to the recipientcells in large aggregates. A particle delivery system suitable for usewith the invention is the helium acceleration PDS-1000/He gun, which isavailable from Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.) (Sanfordet al., Technique 3:3–16 (1991)).

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain 1000 or more loci of cellstransiently expressing a marker gene. The number of cells in a focusthat express the exogenous gene product 48 hours post-bombardment oftenranges from one to ten, and average one to three.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small-scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions that influence the physiological state of therecipient cells and which may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example the methods described by Fraley etal., Bio/Technology 3:629–635 (1985) and Rogers et al., Methods Enzymol.153:253–277 (1987). Further, the integration of the Ti-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., Mol. Gen. Genet. 205:34 (1986)).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described in Klee et al., in Plant DNA InfectiousAgents, Hohn and Schell (eds.), Springer-Verlag, New York, pp. 179–203(1985). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide-coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes (Rogerset al., Methods Enzymol. 153:253–277 (1987)). In addition, Agrobacteriumcontaining both armed and disarmed Ti genes can be used for thetransformations. In those plant strains where Agrobacterium-mediatedtransformation is efficient, it is the method of choice because of thefacile and defined nature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene. Morepreferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant, a transgenic plant that contains a singleadded gene, germinating some of the seed produced and analyzing theresulting plants produced for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating, exogenous constructs. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes thatencode a polypeptide of interest. Backcrossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation and combinations of these treatments (See, e.g.,Potrykus et al., Mol. Gen. Genet. 205:193–200 (1986); Lorz et al., Mol.Gen. Genet. 199:178 (1985); Fromm et al., Nature 319:791 (1986);Uchimiya et al., Mol. Gen. Genet. 204:204 (1986); Marcotte et al.,Nature 335:454–457 (1988)). Application of these systems to differentplant strains depends upon the ability to regenerate that particularplant strain from protoplasts. Illustrative methods for the regenerationof cereals from protoplasts are described (Fujimura et al., Plant TissueCulture Letters 2:74 (1985); Toriyama et al., Theor. Appl. Genet. 205:34(1986); Yamada et al., Plant Cell Rep. 4:85 (1986); Abdullah et al.,Biotechnology 4:1087 (1986)).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, Bio/Technology6:397 (1988)). In addition, “particle gun” or high-velocitymicroprojectile technology can be utilized (Vasil et al., Bio/Technology10:667 (1992)). Using the latter technology, DNA is carried through thecell wall and into the cytoplasm on the surface of small metal particlesas described (Klein et al., Nature 328:70 (1987); Klein et al., Proc.Natl. Acad. Sci. USA 85:8502–8505 (1988); McCabe et al., Bio/Technology6:923 (1988)). The metal particles penetrate through several layers ofcells and thus allow the transformation of cells within tissue explants.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens and obtaining transgenic plants have been published forcotton (U.S. Pat. Nos. 5,004,863; 5,159,135; 5,518,908); soybean (U.S.Pat. Nos. 5,569,834; 5,416,011; McCabe et al., Biotechnology 6:923(1988); Christou et al., Plant Physiol. 87:671–674 (1988)); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep.15:653–657 (1996), McKently et al., Plant Cell Rep. 14:699–703 (1995));papaya; pea (Grant et al., Plant Cell Rep. 15:254–258 (1995)); andArabidopsis thaliana (Bechtold et al., C.R. Acad. Sci. Paris, Life Sci.316:1194–1199 (1993)). The latter method for transforming Arabidopsisthaliana is commonly called “dipping” or vacuum infiltration orgermplasm transformation.

Transformation of monocotyledons using electroporation, particlebombardment and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354 (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); corn (Rhodes et al., Science 240:204(1988); Gordon-Kamm et al., Plant Cell 2:603–618 (1990); Fromm et al.,Bio/Technology 8:833 (1990); Koziel et al., Bio/Technology 11:194(1993); Armstrong et al., Crop Science 35:550–557 (1995)); oat (Somerset al., Bio/Technology 10:1589 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., Theor Appl. Genet.205:34 (1986); Part et al., Plant Mol. Biol. 32:1135–1148 (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133–141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al., Plant Cell Rep.7:379 (1988); Battraw and Hall, Plant Sci. 86:191–202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., Bio/Technology 10:691 (1992)) and wheat (Vasilet al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol (PEG) treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454–457 (1988); Marcotte et al., Plant Cell 1:523–532 (1989);McCarty et al., Cell 66:895–905 (1991); Hattori et al., Genes Dev.6:609–618 (1992); Goff et al., EMBO J. 9:2517–2522 (1990)). Transientexpression systems may be used to functionally dissect gene constructs(see generally, Maliga et al., Methods in Plant Molecular Biology, ColdSpring Harbor Press (1995)).

Any of the nucleic acid molecules of the invention may be introducedinto a plant cell in a permanent or transient manner. A nucleic acidmolecule of the present invention may be stably integrated into anuclear, chloroplast or mitochondrial genome, preferably into thenuclear genome.

Other methods of cell or organism transformation can also be used andinclude but are not limited to introduction of DNA into plants by directDNA transfer into pollen (Hess et al., Intern Rev. Cytol. 107:367(1987); Luo et al., Plant Mol Biol. Reporter 6:165 (1988)), by directinjection of DNA into reproductive organs of a plant (Pena et al.,Nature 325:274 (1987)), by direct microinjection of DNA into protoplasts(Crossway et al., Mol. Gen. Genet. 202: 179–185 (1986)), or by directinjection of DNA into the cells of immature embryos followed by therehydration of desiccated embryos (Neuhaus et al., Theor. Appl. Genet.75:30 (1987)). See also EP 0 238 023; Yelton et al., Proc. Natl. Acad.Sci. (U.S.A.), 81:1470–1474 (1984); Malardier et al., Gene, 78:147–156(1989); Becker and Guarente, In: Abelson and Simon (eds.), Guide toYeast Genetics and Molecular Biology, Method Enzymol., Vol. 194, pp.182–187, Academic Press, Inc., New York; Ito et al., J. Bacteriology,153:163 (1983); Hinnen et al., Proc. Natl. Acad. Sci. (U.S.A.), 75:1920(1978); and Bennett and LaSure (eds.), More Gene Manipualtionins infungi, Academic Press, CA (1991).

The regeneration, development and cultivation of plants from singleplant protoplast transformants or from various transformed explants arewell known in the art (Weissbach and Weissbach, In Methods for PlantMolecular Biology, Academic Press, San Diego, Calif., (1988)). Thisregeneration and growth process typically includes the steps ofselection of transformed cells and culturing those individualized cellsthrough the usual stages of embryonic development and through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

The development or regeneration of plants containing a foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of theinvention containing a desired polypeptide is cultivated using methodswell known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

The present invention also provides for the generation of parts of theplants, particularly reproductive or storage parts. Plant parts, withoutlimitation, include seeds, endosperm, ovule, pollen, roots, tubers,stems, leaves, stalks, fruit, berries, nuts, bark, pods, and flowers. Ina particularly preferred embodiment of the present invention, the plantpart is a seed.

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, protein, or oil preparation. Aparticularly preferred plant part for this purpose is a seed. In apreferred embodiment, the feed, meal, protein or oil preparation isdesigned for livestock animals or humans, or both. Methods to producefeed, meal, protein and oil preparations are known in the art. See, forexample, U.S. Pat. Nos. 4,957,748, 5,100,679, 5,219,596, 5,936,069,6,005,076, 6,146,669, and 6,156,227. In a preferred embodiment, theprotein preparation is a high protein preparation. Such a high proteinpreparation preferably has a protein content of greater than 5% w/v,more preferably 10% w/v, and even more preferably 15% w/v. In apreferred oil preparation, the oil preparation is a high oil preparationwith an oil content derived from a plant or part thereof of the presentinvention of greater than 5% w/v, more preferably 10% w/v, and even morepreferably 15% w/v. In a preferred embodiment, the oil preparation is aliquid. In a preferred embodiment, the oil preparation is of a volumegreater than 1, 5, 10 or 50 liters. The present invention provides foroil produced from plants of the present invention or generated by amethod of the present invention. Such oil may exhibit enhanced oxidativestability. Also, such oil may be a minor or major component of anyresultant product. Moreover, such oil may be blended with other oils. Ina preferred embodiment, the oil produced from plants of the presentinvention or generated by a method of the present invention constitutesgreater than 0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% by volume or weightof the oil component of any composition. In another embodiment, the oilpreparation may be blended and can constitute greater than 10%, 25%,35%, 50% or 75% of the blend by volume. Oil produced from a plant of thepresent invention can be admixed with one or more organic solvents orpetroleum distillates.

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). Selected, non-limiting approaches, for breedingthe plants of the present invention are set forth below. A breedingprogram can be enhanced using marker-assisted selection of the progenyof any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Factorssuch as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed set, seedsize, seed density, standability, and threshability will generallydictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredembodiment, a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated selfing and selection, producing many new genetic combinations.

The development of new cultivars requires the development and selectionof varieties, the crossing of these varieties and the selection ofsuperior hybrid crosses. The hybrid seed can be produced by manualcrosses between selected male-fertile parents or by using male sterilitysystems. Hybrids are selected for certain single gene traits such as podcolor, flower color, seed yield, pubescence color, or herbicideresistance, which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. A F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals from thebest families is carried out. Replicated testing of families can beginin the F₄ generation to improve the effectiveness of selection fortraits with low heritability. At an advanced stage of inbreeding (i.e.,F₆ and F₇), the best lines or mixtures of phenotypically similar linesare tested for potential release as new cultivars.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or more podsfrom each plant in a population and thresh them together to form a bulk.Part of the bulk is used to plant the next generation and part is put inreserve. The procedure has been referred to as modified single-seeddescent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seed of apopulation each generation of inbreeding.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g. Fehr, Principles of Cultivar Development Vol. 1, pp. 2–3(1987)).

A transgenic plant of the present invention may also be reproduced usingapomixis. Apomixis is a genetically controlled method of reproduction inplants where the embryo is formed without union of an egg and a sperm.There are three basic types of apomictic reproduction: 1) apospory wherethe embryo develops from a chromosomally unreduced egg in an embryo sacderived from the nucleus, 2) diplospory where the embryo develops froman unreduced egg in an embryo sac derived from the megaspore mothercell, and 3) adventitious embryony where the embryo develops directlyfrom a somatic cell. In most forms of apomixis, pseudogamy orfertilization of the polar nuclei to produce endosperm is necessary forseed viability. In apospory, a nurse cultivar can be used as a pollensource for endosperm formation in seeds. The nurse cultivar does notaffect the genetics of the aposporous apomictic cultivar since theunreduced egg of the cultivar develops parthenogenetically, but makespossible endosperm production. Apomixis is economically important,especially in transgenic plants, because it causes any genotype, nomatter how heterozygous, to breed true. Thus, with apomicticreproduction, heterozygous transgenic plants can maintain their geneticfidelity throughout repeated life cycles. Methods for the production ofapomictic plants are known in the art. See, e.g., U.S. Pat. No.5,811,636.

The following examples are illustrative and not intended to be limitingin any way.

EXAMPLE 1

This example illustrates constructs which were prepared to demonstratethe practice of this invention.

With reference to FIG. 1 there is shown schematically the elements of aDNA construct comprising in series

(a) DNA of a napin promoter,

(b) DNA coding for gamma methyl transferenase (GMT) isolated fromGossypium hirsutium (cotton),

(c) sense oriented DNA of the 3′ UTR of Arabidopsis thaliana fad2,

(d) DNA of an intron in the Arabidopsis thaliana fad2 with splice sitesremoved,

(e) the complement of the (c) element, i.e. the antisense oriented DNAof the 3′UTR of Arabidopsis thaliana fad2, and

(f) DNA of a napin 3′ terminator.

The construct was inserted together with a BAR marker element into avector between TI borders from Agrobacterium tumefaciens. With referenceto SEQ ID NO: 5 the pertinent DNA elements of a vector, which wasdesignated pMON75565, are described in Table 1.

TABLE 1 Elements of vector pMON75565 Bases description of DNA segment 1–285 Agrobacterium tumefaciens right border  520–2282 napin promoter2344–3381 Gossypium hirsutium gmt 3425–3470 napin 3′ transcriptionterminator 3545–3678 fad2 3′ UTR in sense orientation 3687–4818 fad2intron 4823–4947 fad2 3′ UTR in antisense orientation 4985–6199 napin 3′transcription terminator 6381–6780 CaMV 35S promoter 6781–7328 BARmarker gene 7333–7590 NOS transcription terminator 7597–8179Agrobacterium tumefaciens left border

With reference to FIG. 2 there is shown schematically the elements of aDNA construct comprising in series

(a) DNA of a napin promoter,

(b) DNA coding for gamma methyl transferenase (GMT) isolated fromGossypium hirsutium (cotton),

(c) DNA of an intron in the Arabidopsis thaliana fad2 with splice sitesremoved, and

(d) DNA of a napin 3′ terminator.

The construct was inserted together with a BAR marker element into avector between TI borders from Agrobacterium tumefaciens. With referenceto SEQ ID NO: 6 the pertinent DNA elements of a vector, which wasdesignated pMON75571, are described in Table 2.

TABLE 2 Elements of vector pMON75571 Bases description of DNA segment 1–285 Agrobacterium tumefaciens right border  520–2282 napin promoter2344–3381 Gossypium hirsutium gmt 3396–4515 fad2 intron 4519–5733 napin3′ transcription terminator 5915–6314 CaMV 35S promoter 6315–6862 BARmarker gene 6867–7124 NOS transcription terminator 7131–7713Agrobacterium tumefaciens left border

Transformation of Plants with pMON75565 and pMON75571

Vectors, pMON75565 and pMON75571, are used in Arabidopsis thaliana planttransformation to direct the expression of GMT and inhibit theexpression of the fad2 gene. Binary vector constructs pMON75565 andpMON75571 are transformed into ABI strain Agrobacterium cells by themethod of Holsters et al., Mol. Gen. Genet. 163:181–187 (1978).Transgenic Arabidopsis thaliana plants are obtained byAgrobacterium-mediated transformation as described by Valverkens et al.,Proc. Nat. Acad. Sci. USA 85:5536–5540 (1988), Bent et al., Science265:1856–1860 (1994), and Bechtold et al., C.R. Acad. Sci., LifeSciences 316:1194–1199 (1993). Transgenic plants are selected bysprinkling the transformed R₁ seeds directly onto soil and thenvernalizing them at 4° C. in the absence of light for 4 days. The seedsare then transferred to 21° C., 16 hours light and sprayed with a 1:200dilution of Finale (Basta) herbicide at 7 days and 14 days afterseeding. Transformed plants are grown to maturity and the R₂ seed thatis produced is analyzed for tocopherol content.

FIGS. 3A and 3B show data from the alpha-tocopherol level analysis fromR₂ seed of transgenic Arabidopsis thaliana plants expressing GMTs frompMON75565 (top) or pMON75571 (bottom) under the control of the napinseed-specific promoter. Table 3 below gives specific tocopherol levelresults (alpha, gamma and delta) for various transformed and controlplant lines.

TABLE 3 alpha gamma delta Construct Toco Toco Toco total Toco % alphaGeneration Control 7 453 12 472 1.5 R3 9 446 12 467 1.9 R3 5 440 10 4551.1 R3 7 460 12 479 1.5 R3 9 460 13 482 1.9 R3 6 443 10 459 1.3 R3 6 45911 476 1.3 R3 8 456 10 474 1.7 R3 6 447 11 464 1.3 R3 7 436 9 452 1.5 R3pMON 67 386 11 464 14.4 R2 75565 320 152 5 477 67.1 R2 304 142 6 45267.3 R2 309 142 5 456 67.8 R2 292 134 4 430 67.9 R2 320 143 5 468 68.4R2 360 145 5 510 70.6 R2 317 121 4 442 71.7 R2 329 124 4 457 72.0 R2 33679 3 418 80.4 R2 369 78 3 450 82.0 R2 392 68 4 464 84.5 R2 391 66 4 46184.8 R2 422 51 2 475 88.8 R2 pMON 10 492 13 515 1.9 R2 75571 137 350 8495 27.7 R2 296 166 5 467 63.4 R2 313 136 5 454 68.9 R2 364 124 4 49274.0 R2 354 119 3 476 74.4 R2 371 91 2 464 80.0 R2 381 87 2 470 81.1 R2391 52 2 445 87.9 R2 422 55 3 480 87.9 R2 436 54 2 492 88.6 R2 410 45 2457 89.7 R2 449 45 1 495 90.7 R2 439 31 1 471 93.2 R2 475 22 1 498 95.4R2

FIGS. 3A and 3B and Table 3 show that the construct increased the levelof alpha-tocopherol in the transformed plant lines compared withnon-transformed plant lines.

Fatty acid compositions are analyzed using gas chromatography from seedof Arabidopsis lines transformed with constructs pMON75565 andpMON75571. Table 4 provides a summary of fatty acid levels that areobtained using these constructs. As can be seen, the expression thepMON75565 construct results in increased expression of oleic acid(18: 1) and minor decrease in the expression of linoleic acid (18:2) andlinolenic acid (18:3), with virtually no change in the levels of stearicacid (18:0). There are no significant changes in 12:0, 14:0, 16:0, 16:1,20:0, 20:1, 20:2, 22:0, 22:1 and 22:2 fatty acid levels. The results forpMON75571 and pMON75565 differ. Moreover, there is a higher percentageof success using RNAi suppression as compared to sense suppression.

Table 5 provides a summary of oil levels that are obtained using thedescribed constructs. As can be seen, the total levels of protein,carbon, nitrogen and sulfur remain virtually the same when the pMON75565and pMON75571 constructs are used as compared to the control constructs.

FIG. 4 depicts a graphic presentation of both fatty acid and oil levelsthat are obtained using the pMON75565 and pMON75571 constructs. LinesAT_G490 and AT_G499 (both obtained using pMON75565) have the highestoleic acid and exhibit alpha-tocopherol phenotypes and are both takenonto the next generation for tocopherol and oleic acid and oil analysis.Expression of the double-stranded FAD2 RNA sequences result in themodification of both the fatty acid and the oil compositions.

In order to confirm the phenotype of the pMON75565 construct, the R₂plants expressing the pMON75565 construct are self crossed to obtain R₃plants. Table 6 confirms that the expression of the double-stranded FAD2RNA sequences by the R₃ plants result in the modification of both thefatty acid and the oil compositions. Specifically, the levels of oleicacid are increased as compared to the control construct, and the levelsof linoleic and linolenic acid are slightly decreased. Such a result isconsistent with a down-regulation of FAD2 expression.

Table 7 and FIG. 5 confirm that the R₃ plants express the GMT RNAsequence, which results in increased levels of alpha-tocopherol, whilethe total levels of tocopherol remain essentially the same.

These data show that the constructs of the present invention up-regulatecotton GMT protein and down-regulate the expression of FAD2. Increasedexpression of GMT results in an increase in alpha-tocopherol levels.(GMT converts gamma-tocopherol to alpha-tocopherol). An oleic acid levelincrease and linoleic acid level decrease is consistent with downregulation.

TABLE 4 CONSTRUCT STRAIN ID 18:0 18:1 18:2 18:3 Control 9979-54-49 2.9814.09 28.71 18.88 9979-54-50 2.89 14.28 29.51 18.41 9979-54-51 2.8 14.4629.28 18.43 9979-54-52 2.75 15.5 29.53 17.57 9979-54-53 2.78 15.61 29.3917.63 pMON AT_G485 3.04 22.4 20.82 18.38 75565 AT_G486 2.9 18.09 25.8818.25 AT_G487 2.95 16.39 26.28 19.71 AT_G488 2.97 22.53 20.95 18.16AT_G489 2.8 28.87 18.17 15.53 AT_G490 3 32.34 15.18 15.05 AT_G492 2.818.26 26.68 17.51 AT_G493 2.86 24.25 21.16 16.85 AT_G494 3.02 23.3620.44 18.12 AT_G495 2.9 23.9 21.43 16.88 AT_G496 3.02 21.53 22.08 18.59AT_G497 2.79 27.9 17.46 16.58 AT_G498 2.88 19.35 24.42 18.22 AT_G4993.04 30.19 17.08 15.55 Control 9979-54-59 2.84 14.86 29.6 17.919979-54-60 2.83 14.96 29.41 18.14 9979-54-61 3.02 14.97 29.05 18.629979-54-62 2.71 14.78 29.6 18.18 9979-54-63 2.95 15.29 30.13 17.43 pMONAT_G500 2.84 15.38 28.74 18.39 75571 AT_G501 2.75 16.73 29.31 16.88AT_G502 2.85 15.86 27.86 18.79 AT_G503 2.8 17.18 29.52 16.38 AT_G504 2.915.29 29.01 18.38 AT_G505 2.93 16.25 28.94 17.59 AT_G506 2.86 16.3 29.1817.23 AT_G507 2.89 16.31 27.88 18.27 AT_G508 2.98 16.44 29.93 16.73AT_G509 2.89 15.77 28.8 17.9 AT_G510 2.84 16.91 29.78 16.44 AT_G511 2.7915.32 27.82 19.05 AT_G512 2.77 17.88 29.68 15.62 AT_G513 2.86 16.7 29.5216.78 AT_G514 2.86 15.84 28.66 18.19

TABLE 5 CONSTRUCT EVENT GENERATION % OIL % PRO % C % N % S COLOR Control9979-AT00002-54-49 R3 36.4 22.3 53.4 3.7 0.75 0.981 9979-AT00002-54-50R3 35.4 22.7 52.8 3.8 0.86 0.985 9979-AT00002-54-51 R3 35.1 23.5 53 3.90.88 0.974 9979-AT00002-54-52 R3 37.3 21.5 53.6 3.6 0.85 0.9789979-AT00002-54-53 R3 35.4 23.5 53 3.9 1.03 0.968 pMON AT_G485 R2 3225.2 51.8 4.2 0.89 0.982 75565 AT_G486 R2 36.9 22.6 53.8 3.8 0.79 0.981AT_G487 R2 35.7 23.1 53.1 3.8 0.86 0.98 AT_G488 R2 36.9 22.5 53.9 3.80.74 0.979 AT_G489 R2 37.1 22.2 53.9 3.7 0.91 0.984 AT_G490 R2 37.2 2254 3.7 0.86 0.981 AT_G492 R2 36.8 21.7 53.4 3.6 0.89 0.986 AT_G493 R237.2 22.8 53.9 3.8 0.97 0.976 AT_G494 R2 36.8 22.3 53.7 3.7 0.8 0.975AT_G495 R2 36.3 21.7 53.5 3.6 0.9 0.999 AT_G496 R2 36.5 23 53.6 3.8 0.80.984 AT_G497 R2 35.5 23.5 53.2 3.9 0.95 0.983 AT_G498 R2 37.1 22.9 53.83.8 0.91 0.988 AT_G499 R2 36.5 22.4 53.6 3.7 0.83 0.985 Control9979-AT00002-54-59 R3 36.5 22.5 53.7 3.8 0.96 0.977 9979-AT00002-54-60R3 36.3 22.4 53.6 3.7 0.96 0.978 9979-AT00002-54-61 R3 35.9 23 53.5 3.80.94 0.976 9979-AT00002-54-62 R3 36.3 22.9 53.6 3.8 1 0.9779979-AT00002-54-63 R3 36 22.9 53.6 3.8 0.95 0.975 pMON AT_G500 R2 37.122.5 53.9 3.7 0.94 0.976 75571 AT_G501 R2 36.2 22.9 53.5 3.8 1.14 0.971AT_G502 R2 36.3 23.4 53.7 3.9 1.01 0.976 AT_G503 R2 36.2 22.2 53.6 3.7 10.98 AT_G504 R2 37.1 22.1 53.9 3.7 0.96 0.974 AT_G505 R2 37.4 21.7 543.6 0.88 0.983 AT_G506 R2 38 21.3 54.3 3.6 0.95 0.976 AT_G507 R2 36.523.1 53.7 3.8 1.01 0.974 AT_G508 R2 36.9 22.2 53.8 3.7 0.97 0.981AT_G509 R2 36.7 22.3 53.7 3.7 0.99 0.978 AT_G510 R2 36.9 22.2 53.9 3.70.98 0.978 AT_G511 R2 34.8 23.8 53 4 1 0.982 AT_G512 R2 35 23.7 53.2 3.91.15 0.973 AT_G513 R2 36.1 22.6 53.4 3.8 0.99 0.982 AT_G514 R2 37.3 22.354 3.7 0.96 0.976

TABLE 6 CONSTRUCT STRAIN ID 18:0 18:1 18:2 18:3 pMON AT_G490-2 2.95 21.423.91 17.38 75565 AT_G490-4 2.99 22.46 22.47 17 AT_G490-3 2.83 22.7822.64 17.13 AT_G490-8 2.88 22.82 22.81 16.59 AT_G490-5 3 23.33 22.5116.51 AT_G490-6 2.93 26.1 20.29 16.02 AT_G490-7 3.07 27 19.72 15.89AT_G490-9 2.99 28.59 18.55 15.59 AT_G490-1 2.94 29.9 18.12 14.83AT_G490-10 2.99 31.8 15.49 14.59 AT_G499-9 3.25 26.35 20.47 16.09AT_G499-1 3.12 27.19 17.99 16.59 AT_G499-6 3.13 28.49 20.52 14.81AT_G499-2 3.05 28.86 19.75 14.73 AT_G499-3 3.11 30.21 18.27 14.88AT_G499-5 3.11 30.76 19.83 13.71 AT_G499-10 3.09 32.56 15.77 14.33AT_G499-8 2.91 32.88 16.02 14.46 AT_G499-4 2.86 33.16 16.08 14.17AT_G499-7 3.67 34.04 14.53 11.07 Control 9979-40-92 2.74 15.3 29.0717.16 9979-40-94 2.64 15.9 29.02 17.16 9979-40-95 2.81 15.92 29.03 17.359979-40-88 2.85 16.17 28.87 17.14 9979-40-97 2.79 16.42 28.9 16.589979-40-90 2.56 16.5 29.15 16.45 9979-40-93 2.72 16.65 29.22 16.319979-40-91 2.67 16.84 29.61 16.33 9979-40-96 2.78 16.88 29.07 16.449979-40-89 2.71 16.92 28.88 16.51 9979-40-100 2.67 14.86 28.84 17.599979-40-105 2.81 15.08 28.3 18 9979-40-99 2.78 15.4 28.78 17.719979-40-101 2.73 15.6 28.74 17.44 9979-40-103 2.85 15.67 29.09 17.349979-40-106 2.69 15.83 28.96 17.31 9979-40-102 2.87 15.94 28.45 17.259979-40-107 2.79 16.75 29.16 16.4 9979-40-104 2.82 16.78 28.41 17.039979-40-98 2.89 16.89 27.99 16.94

TABLE 7 alpha- gamma- delta- Total % alpha- Construct Strain ID TocoToco Toco Toco Toco Generation Control 9979-40-100 5 495 16 516 1 R39979-40-94 5 469 15 489 1 R3 9979-40-93 6 468 14 488 1 R3 9979-40-101 6461 14 481 1 R3 9979-40-95 6 455 14 475 1 R3 9979-40-91 7 491 17 515 1R3 9979-40-90 7 491 16 514 1 R3 9979-40-96 7 490 15 512 1 R3 9979-40-997 473 16 496 1 R3 9979-40-106 7 471 15 493 1 R3 9979-40-107 7 469 14 4901 R3 9979-40-103 7 458 14 479 1 R3 9979-40-92 7 447 15 469 1 R39979-40-89 8 498 18 524 2 R3 9979-40-88 8 496 16 520 2 R3 9979-40-102 8485 15 508 2 R3 9979-40-97 8 474 16 498 2 R3 9979-40-98 9 462 14 485 2R3 9979-40-104 9 460 15 484 2 R3 9979-40-105 9 453 15 477 2 R3 pMON75565AT_G499-9. 286 161 7 454 63 R3 AT_G490-8. 268 143 8 419 64 R3 AT_G499-5.274 147 7 428 64 R3 AT_G490-4. 291 153 7 451 65 R3 AT_G490-2. 282 143 7432 65 R3 AT_G499-2. 286 145 7 438 65 R3 AT_G499-6. 301 152 7 460 65 R3AT_G490-5. 274 123 8 405 68 R3 AT_G490-3. 285 128 8 421 68 R3 AT_G490-9.312 116 7 435 72 R3 AT_G490-7. 330 85 6 421 78 R3 AT_G490-10. 330 80 6416 79 R3 AT_G499-3. 352 84 6 442 80 R3 AT_G499-1. 344 71 5 420 82 R3AT_G490-1. 368 71 6 445 83 R3 AT_G499-10. 380 56 5 441 86 R3 AT_G499-4.368 55 4 427 86 R3 AT_G499-7. 441 56 4 501 88 R3 AT_G499-8. 423 48 4 47589 R3 AT_G490-6. 367 34 4 405 91 R3

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A nucleic acid molecule comprising a first nucleic acid segmentcomprising a polypeptide encoding sequence and a second nucleic acidsegment comprising a gene suppression sequence, wherein transcription ofsaid nucleic acid molecule in a host cell results in the simultaneousexpression of a polypeptide by said polypeptide encoding sequence andsuppression of a second gene in said host cell, and wherein said firstnucleic acid segment and said second nucleic acid segment are operablylinked to a single promoter in a polycistronic configuration.
 2. Thenucleic acid molecule according to claim 1, wherein said second nucleicacid segment is expressed as a dsRNA molecule.
 3. The nucleic acidmolecule according to claim 2, wherein said second nucleic acid segmenthas at least 21 contiguous nucleotides corresponding to an mRNA.
 4. Thenucleic acid molecule according to claim 3, wherein said second nucleicacid segment has at least 21 contiguous nucleotides corresponding to anintron from said mRNA.
 5. The nucleic acid molecule according to claim3, wherein said second nucleic acid segment has at least 21 contiguousnucleotides corresponding to an exon from said mRNA.
 6. The nucleic acidmolecule according to claim 3, wherein said second nucleic acid segmenthas at least 21 contiguous nucleotides corresponding to a 3′ UTR fromsaid mRNA.
 7. The nucleic acid molecule according to claim 3, whereinsaid second nucleic segment acid has at least 21 contiguous nucleotidescorresponding to a 5′ UTR from said mRNA.
 8. The nucleic acid moleculeaccording to claim 1, wherein said suppression of a gene is suppressionof an endogenous gene to said host cell.
 9. A plant having in its genomea nucleic acid molecule of claim
 1. 10. A method of simultaneouslyaltering the expression of more than one RNA molecule in a plantcomprising introducing into the genome of said plant a nucleic acidmolecule of claim
 1. 11. The method according to claim 10, wherein saidsecond nucleic acid segment is expressed as a dsRNA molecule.
 12. Themethod according to claim 11, wherein said second nucleic acid segmenthas at least 21 contiguous nucleotides corresponding to an mRNA.
 13. Themethod according to claim 11, wherein said second nucleic acid segmenthas at least 21 contiguous nucleotides corresponding to an intron fromsaid mRNA.
 14. The method according to claim 11, wherein said secondnucleic acid segment has at least 21 contiguous nucleotidescorresponding to an exon from said mRNA.
 15. The method according toclaim 11, wherein said second nucleic acid segment has at least 21contiguous nucleotides corresponding to a 3′ UTR from said mRNA.
 16. Themethod according to claim 11, wherein said second nucleic acid segmenthas at least 21 contiguous nucleotides corresponding to a 5′ UTR fromsaid mRNA.
 17. The method according to claim 10, wherein the level ofexpression of at least one of said more than one RNA molecules is atleast partially reduced.
 18. The method according to claim 17, whereinsaid level of expression of at least one of said more than one RNAmolecules is substantially reduced.
 19. The method according claim 18,wherein the level of expression of at least one of said more than oneRNA molecules is effectively eliminated.
 20. A nucleic acid moleculecomprising a first nucleic acid segment comprising a polypeptideencoding sequence and a second nucleic acid segment comprising a genesuppression sequence, wherein transcription of said nucleic acidmolecule in a host cell results in expression of a polypeptide by saidpolypeptide encoding sequence and suppression of a gene in said hostcell, and wherein said first nucleic acid segment and said secondnucleic acid segment are operably linked to a single promoter in apolycistronic configuration, wherein said first and said second nucleicacid segments are obtained from different genes.